A novel CFHR5 mutation associated with C3 glomerulonephritis in a Turkish girl

Nesrin Besbas, Bora Gulhan, Safak Gucer, Emine Korkmaz & Fatih Ozaltin

Journal of Nephrology

ISSN 1121-8428 Volume 27 Number 4

J Nephrol (2014) 27:457-460 DOI 10.1007/s40620-013-0008-1

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1 23 Author's personal copy

J Nephrol (2014) 27:457–460 DOI 10.1007/s40620-013-0008-1

CASE REPORT

A novel CFHR5 mutation associated with C3 glomerulonephritis in a Turkish girl

Nesrin Besbas • Bora Gulhan • Safak Gucer • Emine Korkmaz • Fatih Ozaltin

Received: 17 July 2013 / Accepted: 31 July 2013 / Published online: 5 December 2013 Ó Italian Society of Nephrology 2013

Abstract C3 glomerulopathy defines a subgroup of syndrome and persistently low C3 level, thus expanding the membranoproliferative glomerulonephritis (MPGN) char- genetic and phenotypic spectrum of the disease. Ecu- acterized by complement 3 (C3)-positive, immunoglobu- lizumab seems to be ineffective in this subtype. lin-negative deposits in immunofluorescence microscopy. It comprises 3 clinical conditions: dense deposit disease, Keywords C3 glomerulopathy Á CFHR5-related C3 glomerulonephritis, and complement factor H-related 5 nephropathy Á CFHR5 mutation Á Hypocomplementemia Á (CFHR5) nephropathy. Mutations in genes encoding reg- Nephrotic syndrome Á Eculizumab ulatory proteins of the alternative complement pathway have been described. A 16-year-old girl was admitted to the hospital due to periorbital edema. Nephrotic syndrome Introduction accompanied by low C3 level was diagnosed. Renal biopsy showed MPGN in light microscopy, only C3 deposits in The term membranoproliferative glomerulonephritis immunofluorescence microscopy, and subendothelial (MPGN) defines a heterogeneous group of kidney diseases electron dense deposits and capillary basement membrane that frequently lead to kidney failure [1]. Histologically, it thickening with double contour formation in electron is characterized by mesangial interposition and the dupli- microscopy. C3 nephritic factor and anti complement fac- cation of glomerular basement membranes that are asso- tor H antibody were negative. Complement factor H level ciated with immune deposits, all of which give an was normal. Genetic screening showed a novel heterozy- appearance of hyperlobulated glomeruli. Recent advances gous p.Cys269Arg variation in the CFHR5 gene without regarding the pathomechanisms of MPGN have led to a any mutation in CFH and CFI genes. Eculizumab therapy more mechanistic classification based on the presence of was started but was unsuccessful at 10 months of follow- immunoglobulins (Igs) and/or complement 3 (C3) deposits up. We have identified a novel heterozygous variation in in immunofluorescence microscopy. In this context, ‘‘C3 CFHR5-related nephropathy presenting with nephrotic glomerulopathy’’ (C3G) has been proposed for those patients with C3(?)Ig(-) MPGNs [2, 3]. Distinct forms of C3G include dense deposit disease (DDD), C3 glomeru- N. Besbas Á B. Gulhan Á F. Ozaltin (&) lonephritis (C3GN), and complement factor H-related 5 Department of Pediatric Nephrology, Hacettepe University (CFHR5) nephropathy. DDD is characterized by intra- Faculty of Medicine, Ankara, Turkey membranous electron dense deposits, in contrast to C3GN e-mail: [email protected] which has subendothelial or, rarely, subepithelial and S. Gucer mesangial electron dense deposits. Acquired and genetic Department of Pediatric Pathology, Hacettepe University defects leading to dysregulation of the alternative pathway Faculty of Medicine, Ankara, Turkey (AP) of the complement system are the underlying patho- genetic mechanism [3]. Common to all C3Gs is the E. Korkmaz Á F. Ozaltin Nephrogenetics Laboratory, Hacettepe University Faculty of uncontrolled activity of AP, which can result from muta- Medicine, Ankara, Turkey tions in genes encoding complement proteins such as CFH, 123 Author's personal copy

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CFI, C3, CFB or acquired antibodies stabilizing C3 con- and lambda light chains (Fig. 1b). Electron microscopy vertase of AP (i.e. C3 nephritic factor) or inhibiting com- showed subendothelial deposits, and capillary basement plement regulators such as anti factor H antibody [4, 5]. membrane thickening with double contour formation CFHR5 (complement factor H-related protein 5) is a regu- (Fig. 1c). Prednisone (45 mg/day) and enalapril (10 mg/ lator of the alternative complement pathway and has been day) were administered. This treatment failed to induce shown to co-localize in the glomerulus with complement remission and low C3 persisted. Therefore, pulse steroid under pathological conditions [6]. Autosomal dominant (500 mg/day, 3 consecutive days for 3 months) and losar- mutations that lead to C3 glomerulonephritis characterized tan (25 mg/day) were added to the therapy. Proteinuria and by glomerular inflammation with complement C3 but not Ig low C3 level did not resolve. Then, eculizumab (900 mg/ or C1q deposits in the kidney have been recently identified week for 4 weeks, 1,200 mg/every 2 weeks thereafter) was [7]. In the original report, the index patient and affected started. After 10-month follow-up, proteinuria (3 g/day) relatives with the disease presented with microscopic with low C3 (20 mg/dl) still continued; however, serum hematuria as well as episodes of macroscopic hematuria albumin had returned to normal (3.3 g/dl) with normal following upper respiratory tract infections, whereas sub- serum creatinine (0.49 mg/dl). sequent studies showed association of CFHR5 mutations Genetic screening detected a novel heterozygous mis- with proteinuria, thus expanding the spectrum [8]. sense variation (c.805T[C; p.Cys269Arg) that was absent Here, we report a pediatric patient with C3G presenting in 180 healthy individuals in gene-encoding CFHR5 with nephrotic syndrome without hematuria that was (Fig. 1d). No mutation was detected in the gene encoding associated with a novel CFHR5 mutation in whom ecu- complement factor H and factor I except for a homozygous lizumab was unsuccessful. known CFI polymorphism (rs11098044).

Case report Discussion

A 16-year-old girl was admitted to the hospital due to We report an adolescent girl who presented with nephrotic periorbital edema. Her previous and family histories were syndrome and persistently low C3 level associated with an unremarkable. Laboratory parameters were as follows: MPGN pattern in light microscopy of kidney biopsy. We hemoglobin 12.4 g/dl, platelet count 338 9 109/l, white diagnosed C3G given the C3(?)Ig(-) staining pattern in blood cell count 9.600/ll, blood urea nitrogen 8.3 mg/dl immunofluorescence microscopy and further classified the (normal range 5–18 mg/dl), serum creatinine 0.53 mg/dl patient as CFHR5-related nephropathy due to a mutation in (normal range 0.5–0.9 mg/dl), serum albumin 2.69 g/dl CFHR5 [7]. The functional effect of the p.Cys269Arg (normal range 3.2–4.5 g/dl). Serum electrolytes were variation in our patient was evaluated by PolyPhen2 web within normal limits. Serum levels of C3 and C4 were tool with a score result of 1.0 suggesting that this variation 16 mg/dl (normal range 79–152 mg/dl) and 13.3 mg/dl was a damaging one [9]. Cysteine at position 269 is of (normal range 10–40 mg/dl), respectively, with normal critical importance in making a disulfide bond in the ter- plasma levels of complement factors H and I. Urinalysis tiary protein structure; therefore, the cysteine to arginine showed proteinuria (300 mg/dl) with a normal sediment exchange is predicted to be deleterious (http://www.uni and a specific gravity of 1,014. Daily urinary protein prot.org/uniprot/Q9BXR6). Thus, we think that the excretion was 2.086 mg. Renal ultrasonography showed p.Cys269Arg variation is responsible for the phenotype in increased echogenicity in both kidneys. No anti nuclear, our patient. C3G equally affects all ages and both genders anti dsDNA, anti-complement factor H antibodies or C3 and typically presents with hematuria and proteinuria [10]. nephritic factor were detected. A renal biopsy was per- Functional and genetic studies of the alternative comple- formed. Light microscopy showed a membranoprolifera- ment pathway in these disorders have identified heteroge- tive pattern in 14 glomeruli with varying degrees of neous abnormalities. C3 nephritic factors, factor H mesangial matrix increase, segmental endocapillary pro- autoantibodies and mutations in the genes involved in liferation, thickened capillary walls, and double contour regulation of the alternative complement pathway such as formation. Four glomeruli were segmentally or globally CFH and CFI have been demonstrated in patients [10, 11]. sclerosed. The tubulointerstitial area revealed mild fibrosis, We did not detect C3 nephritic factor or anti-complement focal tubular atrophy, a group of foamy histiocytes and factor H antibody in our patient. However, it is known that patchy infiltration of mononuclear inflammatory cells. The the sensitivity of C3 nephritic factor is relatively low as it vessels were unremarkable (Fig. 1a). Immunofluorescence becomes positive in up to 80 % of DDD and 45 % of C3G studies were strongly positive for C3 and weakly positive [12]. DDD associated with CFHR5 mutation has been for IgM but were negative for IgG, IgA, C1q, C4, kappa reported as well [13]. However, we excluded DDD in our 123 Author's personal copy

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Fig. 1 a Light microscopy shows MPGN with prominent capillary staining for C3 (9400); c electron microscopy shows basement wall thickening, segmental endocapillary proliferation, periglomeru- membrane thickening and small subendothelial deposits (arrows) lar fibrosis, and double contour formation (PAS 9200); b immuno- (912,000); d Sequence electropherogram of the patient shows a fluorescence microscopy shows strong capillary wall and mesangial heterozygous c.805T[C (p.Cys269Arg) variation (arrow) patient owing to the lack of electron dense deposits within same founder mutation. In contrast to the initial report in the glomerular basement membrane (i.e. this is the differ- which hematuria was the main clinical finding, 38 % of entiating feature in electron microscopy). C3G is geneti- patients had in addition proteinuria in the second report of cally heterogeneous. Different mutations have been the same group. Twenty-eight patients with proteinuria reported by different researchers suggesting that C3G developed kidney failure. Of them, 23 (53.5 %) were male results from diverse abnormalities in the AP leading to whereas 5 were female (10.4 %) suggesting a marked subsequent glomerular injury [14]. We found no mutation gender difference in renal prognosis. None of the patients in CFH and CFI. Taken together, these results suggested described hitherto had persistently low C3 level [8]. In our that there might be another abnormality in our patient that patient, however, nephrotic syndrome without hematuria was responsible for the phenotype. CFHR5 nephropathy was the main clinical presentation and a persistently low has been described in a Cypriot family with autosomal C3 level was the remarkable finding. Despite a similar dominant hematuria associated with C3G in kidney biopsy histopathological picture, this apparent phenotypic vari- [7]. CFHR5 is one of the five complement factor H-related ability most likely resulted from different effects of dif- proteins. In the original report, the mutant CFHR5 protein ferent mutations described here and in Cypriot families, arising from a duplication of exons 2–3 of the CFHR5 gene suggesting the presence of genetic heterogeneity in this was shown to bind to surface-bound activated C3b less entity. effectively than the wild type suggesting that the mutant Persistently low C3 level associated with CFHR5 protein might increase C3 convertase activity and promote mutation in our patient suggested dysregulation in the function of the complement system [7]. Athanasiou et al. complement system and prompted us to start eculizumab, a [8] have recently reported histological, molecular and monoclonal IgG antibody that targets C5 and prevents the clinical findings of 91 patients from 16 families with the generation of C5b and membrane attack complex, C5b-9.

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Eculizumab has been approved for treatment of atypical which shares common genetic risk factors with haemolytic hemolytic uremic syndrome (aHUS) and paroxysmal noc- uraemic syndrome. J Med Genet 44(3):193–199 5. Sethi S, Fervenza FC, Zhang Y et al (2011) Proliferative glo- turnal hemoglobinuria (PNH), both of which are disorders merulonephritis secondary to dysfunction of the alternative of the complement system. However, this treatment did not pathway of complement. Clin J Am Soc Nephrol 6(5):1009–1017 yield any beneficial effect in our patient. There are con- 6. Murphy B, Georgiou T, Machet D, Hill P, McRae J (2002) Factor flicting data regarding the efficacy of eculizumab in this H-related protein-5: a novel component of human glomerular immune deposits. Am J Kidney Dis 39(1):24–27 indication [15]. This could be partly explained by the fact 7. Gale DP, de Jorge EG, Cook HT et al (2010) Identification of a that the pathophysiological basis of C3G is more complex mutation in complement factor H-related protein 5 in patients of than that of either aHUS or PNH and that a more proximal Cypriot origin with glomerulonephritis. Lancet 376(9743): level of the AP than that targeted by eculizumab is dis- 794–801 8. Athanasiou Y, Voskarides K, Gale DP et al (2011) Familial C3 turbed. Therefore, prospective randomized controlled glomerulopathy associated with CFHR5 mutations: clinical studies are warranted to assess the efficacy of eculizumab characteristics of 91 patients in 16 pedigrees. Clin J Am Soc in C3G. Additional anticomplement therapies controlling Nephrol 6(6):1436–1446 AP at the C3 convertase level may be needed to treat these 9. Adzhubei IA, Schmidt S, Peshkin L et al (2010) A method and a server for predicting damaging missense mutations. Nat Methods disorders. 7(4):248–249 10. Wada T, Nangaku M (2013) Novel roles of complement in renal Acknowledgments The study was approved by the Hacettepe diseases and their therapeutic consequences. Kidney Int. doi:10. University Ethics Committee (FON 10/03-22) and the parents gave 1038/ki.2013.134 their informed consent. The Nephrogenetics Laboratory was set up by 11. Barbour TD, Pickering MC, Cook HT (2013) Recent insights into the Hacettepe University Infrastructure Project (06A101008). This C3 glomerulopathy. Nephrol Dial Transpl. doi:10.1093/ndt/ study was supported by The Scientific Research and Development gfs430 Office of the Hacettepe University (010A101009). 12. Servais A, Noe¨l LH, Fre´meaux-Bacchi V, Lesavre P (2013) C3 glomerulopathy. Contrib Nephrol 181:185–193 13. Abrera-Abeleda MA, Nishimura C, Smith JL et al (2006) Vari- References ations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranopro- liferative glomerulonephritis type II (dense deposit disease). 1. Braun MC, Licht C, Strife CF (2009) Membranoproliferative J Med Genet 43(7):582–589 glomerulonephritis. In: Avner ED, Harmon WE, Niaudet P, 14. Servais A, Noe¨l LH, Roumenina LT et al (2012) Acquired and Yoshikawa N (eds) Pediatric nephrology, 6th edn. Springer, genetic complement abnormalities play a critical role in dense Berlin, pp 783–797 deposit disease and other C3 glomerulopathies. Kidney Int ´ ¨ 2. Fakhouri F, Fremeaux-Bacchi V, Noel LH, Cook HT, Pickering 82(4):454–464 MC (2010) C3 glomerulopathy: a new classification. Nat Rev 15. Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fre´meaux-Bacchi Nephrol 6(8):494–499 V (2012) French Study Group for aHUS/C3G. Use of eculizumab 3. Sethi S, Fervenza FC (2012) Membranoproliferative glomerulo- for atypical haemolytic uraemic syndrome and C3 glomerulopa- nephritis–a new look at an old entity. N Engl J Med 366(12): thies. Nat Rev Nephrol 8(11):643–657 1119–1131 4. Servais A, Fre´meaux-Bacchi V, Lequintrec M et al (2007) Pri- mary glomerulonephritis with isolated C3 deposits: a new entity

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